Expression of microRNA21 in human periodontal ligament cells osteogenic differentiation

Abstract

Objective To investigate the expression of microRNA21 (miR21) and explore its function and possible mechanism during osteogenic differentiation in human periodontal ligament cells (PDLCs) . Methods PDLCs were isolated from human periodontal ligament tissue and immuncytochemical staining was used to identify their origin. Cells from the 3rd to 4th passage were cultivated in mineralizing medium for 7 days, 14 days and 21 days respectively. Alkaline phosphatase (ALP) experiment and alizarin red (AR) staining were used to identify osteogenic differentiation capacity of PDLCs. The cells were cultured for 4 hours, 1 day, 3 days and 7 days respectively. Expression of miR21, Sprouty 2 (SPRY2) and Runx2 were tested by quantitative real-time PCR and the phosphorylated ERK1/2, phosphorylated p38, SPRY2 and Runx2 were measured by western blot. Cells without osteogenic induction served as controls. Results The cultured PDLCs were derived from mesenchyma. Compared to the control groups, AR staining showed obvious mineralization nodules, and ALP activities were significantly higher in 7 d-, 14 d- and 21 d-osteogenic induction groups (P < 0.05) . MiR21 started up-regulate since 3rd day, and remained high expressed level until 7 days in osteogenic induction groups. Expression of SPRY2 reversed to which of miR21 in the induction groups. The results of western blot showed that both ERK-MAPK and p38 MAPK pathway were activated. ERK-MAPK pathway was activated since the 1st day and sustained during the early osteogenesis period of PDLCs. At the 4th hour of induction, the activity of p38 MAPK was obviously enhanced, then returned to which of the control groups, and up regulated again at 7 days of mineralization cultivation. Conclusions MiR21 was related to osteogenic differentiation of PDLCs and probably modulated ERK-MAPK and p38 MAPK pathways. MiR21 might target SPRY2 as a regulator of ERK-MAPK pathway during osteogenic differentiation of PDLCs. This result can provide basis for further research on its fine mechanism. Key words: MicroRNA21; Periodontal ligament cells; Osteogenic differentiation; Mitogen-activated protein kinases signaling pathway